Continuation of our studies on the chain of events which leads to calcification of growth plate cartilage is proposed. Because of new ultramicro biochemical methodology, we can now examine the interstitial hypertrophic cell cartilage fluid as well as micro-dissected whole cartilage for the presence and type of neutral proteases probably engaged hypothetically in degradation of proteoglycans there, as well as to study the relationship of this process to preliminary disaggregation of PG aggregate inhibitor by cartilage lysozyme. Studies on activation of cartilage proteases or removal of an inhibitor as an alternative will be sought. The relationship of cartilage proteases to the proteases brought in by capillary invasion will be studied. All studies will be made in 5 separate rat preparations which manifest expanded cartilage growth plates. A mineral forming agent already characterized in 2 forms of rickets will be further purified from the top fifth fraction on capillary ultracentrifugation of rachitic Cf1s. Plans for analyzing proteins and phospholipids involved in the nucleational factor are proposed. The extremely high efficiency of the native Cf1 nucleational agent suggests that isolated matrix vesicles or calcifying phospholipids alone may not constitute the total apparatus involved in nucleation. The use of two-dimensional electrophoresis by new methods may indicate association with other proteins. It is possible that some of these proteins are destroyed in two systems used for study, and if this is selective, their additional role in the nucleational complex will be determined. Further evidence to refute or substantiate a role of matrix vesicles as the nucleating agent per se in cartilage should be ascertained. The opening of growth plate functions in the remodelling cartilage of osteoarthritis, a common human disease, make the finding of a regulator which could turn off the growth plate function an eventual goal of clinical importance.